A number of approaches have been described for fermentation of pDNA. The proposed methods differ with regard to the level of control imposed upon the cells and the numerous factors that influence fermentation. Low-level control simply allows plasmid-bearing cells to grow, whereas high-level tightly-controlled fermentations reach high yields of pDNA by specific measures which enhance replication.
For pDNA production on a laboratory scale, cultivation of plasmid-bearing cells in shake flasks is the simplest method, which however normally achieves low yields. Plasmid yields obtained from shake flask cultivations are in the range of 1.5 to 7 mg per L culture broth (O'Kennedy et al., 2003; Reinikainen et al., 1988; O'Kennedy et al., 2000). In shake flask cultivations, several drawbacks such as poor oxygen transfer and the lack of possibility for pH value control, limit the pDNA yield. In U.S. Pat. No. 6,255,099 it was shown that, even in shake flask cultivations, a pDNA yield of up to 109 mg/L can be achieved with certain medium compositions and buffering conditions.
To obtain higher quantities of plasmids, it has been suggested to cultivate the cells in controlled fermenters. A simple fermentation method, in which all nutrients are provided from the beginning and in which no nutrients are added during cultivation, is termed “batch-cultivation” or “batch fermentation”. The application of batch processes in controlled fermenters has led to an increase of pDNA yield per volume. Depending on the plasmid/host combination and on the culture medium, the yield of pDNA obtained from such batch fermentations can vary strongly. Typical plasmid yields reported are in the range between 3.5 and 50 mg/L (O'Kennedy et al., 2003; WO 96/40905; U.S. Pat. No. 5,487,986; WO 02/064752; Lahijani et al., 1996). These cultivations were carried out with culture media containing so-called “complex components” as carbon and nitrogen sources. These components are obtained from biological sources; they include e.g. yeast extract, soy peptone or casein hydrolysate.
Culture media consisting exclusively or predominantly of complex components are termed “complex media”. Media that are composed of both a defined portion (defined carbon source, salts, trace elements, vitamins) and a complex portion (nitrogen source), are termed “semi-defined” media. According to U.S. Pat. No. 5,487,986, a very high amount of various complex components (50 g/L in total) was used.
Culture media containing complex components have the disadvantage that these components originate from biological materials; therefore, the composition of the medium underlies normal natural deviations that make the cultivation process less reproducible. The same applies when a manufacturer changes the production process or when there is a change of supplier. Further disadvantages of using complex medium components are the uncertainty about the exact composition (presence of undesired substances), the impossibility to do stoichiometric yield calculations, the formation of undesired products upon sterilization, difficult handling due to poor dissolution, formation of dust as well as clumping during medium preparation. During fermentation, complex media more readily tend to foaming. Complex components of animal origin (meat extracts, casein hydrolysates) are in particular undesired for pDNA production due to the risk of transmissible spongiform encephalopathy and their use is therefore restricted by pharmaceutical authorities (CBER 1998).
Because of the drawbacks of complex medium components, media have been developed that do not contain any complex components. Such culture media, which are termed “defined” or “synthetic” media, are composed exclusively of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins, and, in view of a possible strain auxotrophy, specific amino acids or other substances such as thiamine. Chemically defined media have the advantage that their composition is exactly known. This allows better process analysis, fermentation monitoring and the specific addition of particular substances which enhance growth or product formation. The well-known composition allows to set up mass balance calculations, which facilitate the prediction of growth and the identification of possibly lacking nutrients. Compared to complex media, fermentations with defined media show enhanced process consistency and improved results during scale-up. Further practical aspects of defined media are better solubility, the absence of inhibiting by-products upon sterilization, and less foam formation during cultivation (Zhang and Greasham, 1999).
Synthetic media, that were not specifically developed for pDNA production, such as M9 (Sambrook and Russel, 2001), may result in a low pDNA yield (WO 02/064752). In batch fermentations with defined culture media that were specifically designed for pDNA production, a higher yield of pDNA was obtained (Wang et al., 2001; WO 02/064752). The latter demonstrated that pDNA homogeneity was more than 90% ccc form. The enhanced yields of pDNA according to WO 02/064752 and Wang et al. (2001) were achieved by supplementation of amino acids that are biosynthetic building blocks of nucleosides, or by the direct addition of nucleosides.
Although batch fermentations are usually simple and short, they have fundamental disadvantages that result in limited plasmid DNA yields. This is due to substrate inhibition and salt precipitation at high nutrient concentrations in the batch medium. Furthermore, the growth rate in batch fermentations cannot be controlled directly; it is therefore unlimited, while steadily changing during fermentation, and ceases only when one or more nutrients are depleted or if metabolic by-products (such as acetate) inhibit growth of the cells.
Consequently, in order to increase biomass and plasmid yield in pDNA production, fed-batch fermentations have been developed. A fed-batch fermentation is a process in which, after a batch phase, a feeding phase takes place in which one or more nutrients are supplied to the culture by feeding.
Different strategies have been pursued for fed-batch fermentation of E. coli to produce plasmid DNA:
One method is the application of a feed-back control algorithm by feeding nutrients in order to control a process parameter at a defined set point. Feed-back control is hence directly related to cell activities throughout fermentation. Control parameters which have been used for feed-back control of fermentations include pH value, on-line measured cell density or dissolved oxygen tension (DOT). These methods have the benefit that high biomass concentrations can be obtained with a reduced risk of overfeeding the culture with the fed nutrient.
For pDNA fermentation, a feed-back algorithm for controlling the dissolved oxygen tension at a defined set point by the feeding rate was used (WO 99/61633). When applying another, more complex algorithm, both the DOT and the pH were used as control parameters for a feed-back cultivation method (U.S. Pat. No. 5,955,323; Chen et al., 1997). In that method, the DOT was controlled by the agitation rate and feeding of a concentrated complex medium (glucose, yeast extract), whereby the pH was concomitantly maintained with ammonium hydroxide.
The application of feed-back algorithms is accompanied by a number of disadvantages. One is, that the feeding rate depends on current process parameters such as the DOT. Irritation of the process due to whatever reason may influence the control parameter and has therefore an impact on the feeding rate and consequently on growth and pDNA yield. For instance, when an antifoam agent has to be added, the DOT changes (normally decreases), which results in a lower feeding rate. This makes the fermentation process less reproducible. Further difficulties arise during scale-up of the process, since fermenters of different geometry or size show different oxygen transfer rates. Since the oxygen transfer rate is coupled to the DOT, the feed-back controlled feeding rates of fermenters of varying size will differ, and therefore the process will not be directly scaleable.
Another disadvantage of feed-back control is that the specific growth rate can not be exactly predefined nor controlled, resulting in suboptimal yields in processes, where the product formation is dependent on growth. However, for pDNA fermentation, a strong dependence of the volumetric and specific plasmid yield on the specific growth rate was shown (WO 96/40905; O'Kennedy et al., 2003).
Control of the specific growth rate can be achieved by another fundamental feeding mode based on the supply of feed medium following an exponential function. The feeding rate is controlled based on a desired specific growth rate P. When a defined medium is applied, growth can be exactly predicted and pre-defined by the calculation of a biomass aliquot X to be formed based on the substrate unit S provided (under consideration of the biomass yield coefficient YX/S).
The invention described in WO 96/40905 uses an exponential fed-batch process for plasmid DNA production and obtains a high yield of biomass (50 g DCW, dry cell weight per L), but reaches a low pDNA yield (18 mg/L; 0.36 mg/g dry cell weight). In another example for exponential feeding, a plasmid yield of 30 mg/L and 6 mg/g DCW was achieved (O'Kennedy et al., 2003). A higher pDNA yield of 220 mg/L was obtained by Lahijani et al. (1996) by combining exponential feeding with temperature-controllable enhancement of plasmid replication. In these examples of exponential feeding, only O'Kennedy et al. (2003) gave details on pDNA homogeneity, which was 50-70% ccc form. Currently, all exponential fed-batch processes, use complex components in both the batch medium and the feed medium.
In summary, the current state of the art in fermentation for manufacturing therapeutic plasmid DNA can be characterized as follows:
Batch fermentations that are widely applied for pDNA production are associated with technological and economical drawbacks. For batch fermentations, complex or semi-defined media are mostly used, resulting in a pDNA yield that ranges between 3.5 and 68 mg per L culture broth. Fed-batch processes that apply feed-back control either use semi-defined media or a complex pre-culture medium followed by a defined medium in the main culture. With feed-back algorithms, plasmid yields between 100 and 230 mg/L can be obtained. Exponentially fed fermentations use semi-defined culture media. The plasmid yield of exponential fermentations is in a broad range between 18 and 220 mg/L. In general, many pDNA fermentation processes suffer from poor homogeneity (i.e. percentage of supercoiled plasmid). Exceptions are fermentations that use a defined medium in the main culture, where a percentage of ccc form over 90% can be obtained.